1. Immunology and Inflammation

Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans

  1. Danika L Hill  Is a corresponding author
  2. Carly E Whyte
  3. Silvia Innocentin
  4. Jia Le Lee
  5. James Dooley
  6. Jiong Wang
  7. Eddie A James
  8. James C Lee
  9. William W Kwok
  10. Martin S Zand
  11. Adrian Liston
  12. Edward J Carr
  13. Michelle A Linterman  Is a corresponding author
  1. Department of Immunology and Pathology, Monash University, Australia
  2. Immunology Program, The Babraham Institute, Babraham Research Campus, United Kingdom
  3. Division of Nephrology, Department of Medicine and Clinical and Translational Science Institute, University of Rochester Medical Center, United States
  4. Benaroya Research Institute at Virginia Mason, Translational Research Program and Tetramer Core Laboratory, United States
  5. Department of Medicine, Cambridge Biomedical Campus, University of Cambridge, United Kingdom
  6. Cambridge Institute of Therapeutic Immunology & Infectious Disease, Jeffrey Cheah Biomedical Centre, Cambridge Biomedical Campus, University of Cambridge, United Kingdom
  7. Benaroya Research Institute at Virginia Mason, Diabetes Program, United States
  8. Department of Medicine, University of Washington, United States
https://doi.org/10.7554/eLife.70554
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Cite this article
  1. Danika L Hill
  2. Carly E Whyte
  3. Silvia Innocentin
  4. Jia Le Lee
  5. James Dooley
  6. Jiong Wang
  7. Eddie A James
  8. James C Lee
  9. William W Kwok
  10. Martin S Zand
  11. Adrian Liston
  12. Edward J Carr
  13. Michelle A Linterman
(2021)
Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans
eLife 10:e70554.
https://doi.org/10.7554/eLife.70554
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8 figures, 1 table and 10 additional files

Figures

Figure 1 with 6 supplements
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Robust haemagglutinin (HA)-specific CD4+ T cells’ response to seasonal influenza vaccination.

(A) Overview of cohort characteristics, vaccination strains, and immune variables measured before (d0), 7 days (d7), and 42 days (d42) after seasonal influenza vaccination. (B) IgG responses to HA …

Figure 1—figure supplement 1
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B cell flow cytometry gating strategy.

Representative flow cytometry plots for B cell immunophenotyping pre-gated on single, live lymphocytes, stained with the panel outlined in Supplementary file 3.

Figure 1—figure supplement 2
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CD4+ T cell flow cytometry gating strategy.

Representative flow cytometry plots for the analysis of CD4+ T cells, stained with the panel outlined in Supplementary file 4. Histograms show representative staining for ICOS expression on d7 …

Figure 1—figure supplement 3
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Haemagglutinin (HA)-specific CD4+ T cell sorting strategy.

Representative flow cytometry plots for the cell sorting of HA-specific Tet+ cells, stained with the panel outlined in Supplementary file 4. Red gates indicate the sorted populations on d0 and/or …

Figure 1—figure supplement 4
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Haemagglutination inhibition (HAI) responses before and after vaccination.

(A) Log2 HAI titres at baseline (d0), d7, and d42 for cohort 1 (n = 16) and (B) cohort 2 (n = 21). (C) Correlation between HAI and A.Cali09 IgG as measured by Luminex assay for cohorts 1 and 2 …

Figure 1—figure supplement 5
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Cytokine and CD4+ T cell variables altered after vaccination in 18–36-year-old individuals.

(A) The concentration of cytokines in serum (log10 pg/mL) at d0 and d7. Variables with significant log2 fold change in Figure 1 shown. (B) The percentage of circulating T follicular helper (cTfh+) …

Figure 1—figure supplement 6
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Percentage of circulating T follicular helper (cTfh) cells that are Tet+ and CXCR3 and CCR6 expression on haemagglutinin (HA)-specific CD4+ T cells.

(A) Representative flow cytometry gating strategy for CXCR5+ PD-1+ cTfh cells on CD4+ CD45RA- T cells, and the proportion of HA-specific Tet+ cells within the CXCR5+ PD-1+ cTfh cell gate. (B) The …

Figure 2 with 1 supplement
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Circulating haemagglutinin (HA)-specific T follicular helper (Tfh) cells correlate with vaccine IgG response.

(A) Overview of correlation analysis between A.Cali09 IgG response at day 42 (minus d0 baseline titre, d42-d0) and immune variables for cohort 1, cohort 2, and both cohorts combined (All). (B) …

Figure 2—figure supplement 1
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Correlations between haemagglutination inhibition (HAI) assay titres and selected immune parameters.

Correlation between vaccine-induced A.Cali09 HAI titres at d42 with selected immune parameters in both cohort 1 and cohort 2 (n = 37). Dot colour corresponds to the cohort (black = cohort 1, grey = …

Figure 3 with 1 supplement
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Haemagglutinin (HA)-specific circulating T follicular helper (cTfh) response to flu vaccination includes recalled and public T cell receptor (TCR) clonotypes.

(A) Overview of cell types and sample sizes that were sequenced d0 and d7 at each cohort. d0 median = 45 cells (6–258); d7 median = 88 cells (5–1000). (B) Representative pie charts of the …

Figure 3—figure supplement 1
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Cell number and clonotype number from T cell sequencing.

(A) The number of cells sorted and sequenced at each time point for each cohort, 18–36-year-old samples only. (B) The number of sequenced cells displayed against the number of TCRB clonotypes …

Figure 4
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Vaccination-induced circulating T follicular helper (Tfh) cells share a common transcriptional signature with lymph node Tfh cells.

(A) Principal component analysis of 1000 genes with the largest variance in sequenced cells from d0 TET+ cells (shown in grey) and d7 TET+ circulating T follicular helper (cTfh) cells (shown in …

Figure 5 with 1 supplement
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Impaired vaccination immune trajectory in older individuals.

(A) Overview of age groups and sample sizes for each cohort. (B) IgG responses to haemagglutinin (HA) proteins from vaccine influenza strains measured by Luminex for each age group. (C) Diffusion …

Figure 5—figure supplement 1
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Age-related differences in cytokines and haemagglutinin (HA)-specific CD4+ T cell parameters.

(A) The serum concentration (log10 pg/mL) of d0 April, d0 TWEAK, and d42 SCF across age groups and cohorts. Variables with consistently significant (padj<0.05) across both cohorts as in Figure 5G …

Figure 6
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Impaired induction of T follicular helper (Tfh) transcriptional programs and aberrant inflammatory signatures in haemagglutinin (HA)-specific circulating T follicular helper (cTfh) cells from older individuals.

(A) Overview of sample numbers and cell types sequenced at d0 and d7 for each cohort and each age group. Dark and light green dots indicate d7 Tet+ cells sequenced from cohort 2 only. (B) Normalised …

Figure 7
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Gene signatures of TNF, IL-2, and inflammation associate with impaired antibody and T follicular helper (Tfh) cell responses to influenza vaccination.

(A) Correlation between circulating T follicular helper (cTfh) Tet+ cells and inflammation, TNF or IL2 gene signatures scores in d7 Tet+cTfh cells. (B) Correlation between A.Cali09 IgG titre …

Figure 8
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Increased IL-2 production impairs T follicular helper (Tfh) cell formation and the germinal centre response.

Assessment of the Tfh cell and germinal centre response in Il2cre/+; Rosa26stop-flox-Il2/+ transgenic mice that do not switch off IL-2 production, and Il2cre/+; Rosa26+/+ control mice 12 days after …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
OtherViability dye and labelling reagentThermo FisherCat#: 65-0865-18Flow cytometry (1:10,000)
AntibodyAnti-human CD14 APC-eF780 clone: 61D3 (mouse monoclonal)Thermo FisherCat#: 47-0149-42Flow cytometry (0.5 μL/million cells)
AntibodyAnti-human CD16 APC-eF780 clone: eBioCB16 (mouse monoclonal)Thermo FisherCat#: 47-0168-42Flow cytometry (0.3 uL/million cells)
AntibodyAnti-human CD3 BV605 clone: UCHT1 (mouse monoclonal)BioLegendCat#: 300460Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CD19 BB515 clone: HIB19 (mouse monoclonal)BD BiosciencesCat#: 564456Flow cytometry(1 μL/million cells)
AntibodyAnti-IgD BV421 clone: IA6-2 (mouse monoclonal)BD BiosciencesCat#: 563813Flow cytometry(1 μL/million cells)
AntibodyAnti-human CD38 APC clone: HIT2 (mouse monoclonal)Thermo FisherCat#: 17-0389-42Flow cytometry (0.5 μL/million cells)
AntibodyAnti-human CD20 PECY7 clone: 2H7 (mouse monoclonal)BioLegendCat#: 302312Flow cytometry (0.5 μL/million cells)
AntibodyAnti-human CD27 BV650 clone: M-T271 (mouse monoclonal)BD BiosciencesCat#: 564894Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CD24 PerCP-eFluor 710 clone: eBioSN3 (mouse monoclonal)Thermo FisherCat#: 46-0247-42Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CD19 APC-eF780 clone: HIB19 (mouse monoclonal)Thermo FisherCat#: 47-0199-42Flow cytometry (0.3 μL/million cells)
AntibodyAnti-human CD3 BUV 395 clone: UCHT1 (mouse monoclonal)Thermo FisherCat#: 563546Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CD45RA BUV737 clone: HI100 (mouse monoclonal)BD BiosciencesCat#: 564442Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CD4 PercpCy5.5 clone: RPA-T4 (mouse monoclonal)BD BiosciencesCat#: 560650Flow cytometry(0.3 μL/million cells)
AntibodyAnti-human CXCR5 BB515 clone: RF8B2 (rat monoclonal)BD BiosciencesCat#: 564624Flow cytometry (0.9 μL/million cells)
AntibodyAnti-human PD1 APC clone: eBioJ105 (mouse monoclonal)BD BiosciencesCat#: 17-2799-42Flow cytometry (1 μL/million cells)
AntibodyAnti-human ICOS biotin clone: ISA-3 (mouse monoclonal)Thermo FisherCat#: 13-9948-82Flow cytometry(1 μL/million cells)
AntibodyAnti-human CXCR3 BV421 clone: 1C6/CXCR3 (mouse monoclonal)Thermo FisherCat#: 562558Flow cytometry(0.5 μL/million cells)
AntibodyAnti-human CCR6 BV786 clone: 11A9 (mouse monoclonal)BD BiosciencesCat#: 563704Flow cytometry(0.5 μL/million cells)
OtherStreptavidin BV650BD BiosciencesCat#: 563855Flow cytometry (0.2 μL/million cells)
AntibodyAnti-mouse CXCR5 clone: L138D7 (rat monoclonal)BioLegendCat#:145511; RRID:AB_2562127Flow cytometry (1:50)
AntibodyAnti-mouse PD-1 clone: 29F.1A12 (rat monoclonal)BioLegendCat#: 135231; RRID:AB_2566158Flow cytometry (1:5000)
AntibodyAnti-mouse Foxp3 clone: FJK-16s (rat monoclonal)eBioscienceCat#: 48-5773-82; RRID:AB_1518812Flow cytometry (1:400)
AntibodyAnti-mouse B220 clone: RA3-6B2 (Rat monoclonal)eBioscienceCat#: 15-0452-82; RRID:AB_468755Flow cytometry (1:500)
AntibodyAnti-mouse Ki67 clone: 16A8 (rat monoclonal)BioLegendCat#: 652420; RRID:AB_2564285Flow cytometry (1:1000)
AntibodyAnti-mouse/human Bcl6 mouse clone: K112-91 (mouse monoclonal)BD BiosciencesCat#: 561525; RRID:AB_10898007BFlow cytometry (1:200)
AntibodyAnti-mouse CD4 clone: GK1.5 (rat monoclonal)BD BiosciencesCat#: 563790; RRID:AB_2738426Flow cytometry (1:400)

Additional files

Supplementary file 1

Immunological variables.

Table of the immunological parameters used in this study

https://cdn.elifesciences.org/articles/70554/elife-70554-supp1-v1.xlsx
Supplementary file 2

Inflammation, TNF, and IL-2 gene signatures.

Table of the gene names used for the inflammation, TNF, and IL-2 gene signatures.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp2-v1.xlsx
Supplementary file 3

Antibody panel for B cells.

Table of the flow cytometry staining panel used for identifying B cell subsets.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp3-v1.xlsx
Supplementary file 4

Antibody panel for T cells.

Table of the flow cytometry staining panel used for identifying T cell subsets.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp4-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/70554/elife-70554-transrepform1-v1.docx
Source data 1

18–36-year-old samples d7 cTfh vs. d0 gene list.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp5-v1.csv
Source data 2

Lymph node germinal centre Tfh gene signature.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp6-v1.csv
Source data 3

d7 Tfh gene set.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp7-v1.csv
Source data 4

d7 vaccination genes.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp8-v1.csv
Source data 5

Ageing d7 cTfh vs. d0 gene list.

https://cdn.elifesciences.org/articles/70554/elife-70554-supp9-v1.csv

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